Opioid drugs have various effects on perception of pain, consciousness, motor control, mood, autonomic function, and can also induce physical dependence. The endogenous opioid system plays an important role in modulating endocrine, cardiovascular, respiratory, gastrointestinal functions, and immune functions. Opioids, either exogenous or endogenous, exert their actions by binding to specific membrane-associated receptors.
Examples of exogenous opioids presently known include, opium, heroin, morphine, codeine, fentanyl, and methadone, to name only a few. Moreover, a family of over 20 endogenous opioid peptides has been identified, wherein the members possess common structural features, including a positive charge juxtaposed with an aromatic ring that is required for interaction with an opioid receptor. It has been determined that most, if not all the endogenous opioid peptides are derived from the proteolytic processing of three precursor proteins, i.e., pro-opiomelanocortin, proenkephalin, and prodynorphin. In addition, a fourth class of endogenous opioids, the endorphins, has been identified (the gene encoding these proteins has not yet been cloned). In the processing of the endogenous opioid precursor proteins, initial cleavages are made by membrane-bound proteases that cut next to pairs of positively charged amino acid residues, and then trimming reactions produce the final endogenous opioids secreted from cells in vivo. Different cell types contain different processing enzymes so that, for example proopiomelanocortin can be processed into different endogenous peptides by different cells. For example, in the anterior lobe of the pituitary gland, only corticotropin (ACTH), β-lipotropin, and β-endorphin are produced. Both pro-enkephalin and pro-dynorphin are similarly processed by specific enzymes in specific cells to yield multiple opioid peptides.
Pharmacological studies have suggested there are numerous classes of opioid receptors which bind to exogenous and endogenous opioids. These classes differ in their affinity for various opioid ligands and in their cellular and organ distribution. Moreover, although the different classes are believed to serve different physiological functions, there is substantial overlap of function, as well as of distribution.
In particular, there are at least three known types of opioid receptors, mu (μ), delta (δ), and kappa (κ), to which morphine, the enkephalins, and the dynorphins can bind. These three opioid receptor types are the sites of action of opioid ligands producing analgesic effects. However, the type of pain inhibited and the secondary functions vary with each receptor type. The mu receptor is generally regarded as primarily associated with pain relief, and drug or other chemical dependence, i.e., addiction and alcoholism.
The human mu opioid receptor, which modulates corticotropin releasing hormone, has been isolated and described in PCT Application WO 95/07983 (Mar. 23, 1995) (SEQ ID NO:1) as well as in Chen, Y., Mestek, A., Hurley, J. A., & Yu, L. (1993) Mol. Pharmacol. 44, 8–12, and Wang, et al., FEBS Letters, (1994)338:217–222. Furthermore, SEQ ID NO:1 can readily be obtained in GENBANK under accession number L25119. The cDNA therefor contains an open reading frame capable of encoding a protein of 400 amino acid residues with 94% sequence similarity to the rat mu opioid receptor. Hydropathy analysis of the deduced protein indicates the presence of seven hydrophobic domains, typical of G-protein-coupled receptors. The N-terminus contains five potential N-linked glycosylation sites which remain conserved between the human and the rat mu opioid receptor. A variant in which Asn-40 is changed to Asp (N40D) is reported in GENBANK Accession No. U12569. New polymorphisms G24A (silent), G779A (Arg260His), and G942A (silent) of the mu opioid receptor have been described in co-pending application Ser. No. 09/113,426, filed Jul. 10, 1998, and Ser. No. 09/351,198, filed Jul. 9, 1999, both of which are incorporated herein by reference in their entireties.
In the body and brain, heroin is biotransformed to morphine, which acts at the mu opioid receptor and results in an euphoric effect and confers the reinforcing properties of the drug and contributes to development of addiction. Heroin addiction can be managed through treatment, primarily methadone maintenance. However, the biological basis of heroin addiction may include diversity of gene structure. Such genetic diversity of the human mu opioid receptor, and the impact of such diversity on receptor function, could contribute to the success or failure of pharmacological management. Similar problems with respect to patient response to pharmacological treatment could occur in most, if not all addictive diseases, such as heroin addiction, alcohol addiction, or cocaine addiction to name only a few, or a combination thereof.
Moreover, addiction to opioid drugs, especially heroin, is a major social problem in the United States, and throughout the world. For example, recent epidemiological assessments sponsored by the NIH-NIDA and other federal agencies have found that around 2.7 million persons in the United States have used heroin at some time. Moreover, the numbers of “hardcore” long-term heroin addicts (addiction being defined herein as self administration of a regular, multiple, daily dose use of a short-acting opioid, such as heroin, for one year or more, with the development of tolerance, physical dependence and drug-seeking behavior, a definition codified in the Federal guidelines governing pharmacotherapy using long-acting agents such as methadone or LAAM, and used as the minimal requirement for entry into treatment) are now estimated to be approximately one million persons. In addition, it has been estimated that around 24 million persons in the United States have used cocaine for some time, and of that number, approximately one million use cocaine regularly, and at least 600,000–700,000 are cocaine addicts.
In view of the importance of the human mu opioid receptor in the study of addiction, and the epidemic proportions of drug addiction, especially to heroin, alcohol or cocaine, or a combination thereof, in the United States and throughout the world, and its involvement in the neuroendocrine system, and physiological functions regulated thereby, efforts have been made to investigate whether any polymorphisms in the gene encoding the human mu opioid receptor exist in the population, and whether such polymorphisms result in a phenotype that has an increased or decreased susceptibility towards development of addiction to exogenous opioids, such as heroin, or alcohol, cocaine, or other addictive drugs. For example, in an article entitled “Human mu opioid receptor gene polymorphisms and vulnerability to substance abuse” (Berrettini, W. H., Hoehe, M. R., Ferraro, T. N., DeMaria, P. A., and Gottheil, E., Addiction Biology 2:303–308 (1997)), two polymorphisms in the human mu opioid receptor gene were reported. One polymorphism (G to T) occurs at nucleotide 175 preceding initiation of translation, and a second coding polymorphism C to T) at nucleotide 229 (with respect to transcription initiation) on exon I results in an Ala to Val residue change. However, data taken from a study indicated the C229T polymorphism does not differ in occurrence with statistical significance in addicts relative to non addicts (idem at 306). No functional studies were reported.
It has been further determined that a receptor for both endogenous and exogenous opioids modulates the activity of the hypothalamus pituitary adrenal axis (HPA) and the hypothalamus pituitary gonadal axis (HPG), which effects the neuroendocrine system and its production of signaling compounds that play important roles in regulation of numerous physiological functions. In particular, the neuroendocrine system involves the integration of the neural and endocrine systems of the body, and is responsible for the coordination of numerous bodily functions. An important part of this system is the hypothalamus, a specialized portion of the brain involved in receiving and relaying messages from the central nervous system to other parts of the body. Upon stimulation by chemical signals from the central nervous system, the hypothalamus secretes hypothalamic hormones, such as corticotropin releasing factor (CRF) or hormone and gonadotropin releasing hormone or luteinizing hormone releasing hormone. These factors in turn stimulate the anterior pituitary gland to secrete tropic hormones, or tropins, which are synthesized as relatively long polypeptides, and then are then biotransformed to produce active peptide hormones. Pro-opiomelanocortin, which is processed into several active peptide hormones, including adrenocorticotropic hormone (ACTH), is an example of a tropic hormone. ACTH stimulates the adrenal cortex to secrete additional hormones, like cortisol, a stress hormone in humans which regulates glucose metabolism, and targets many tissues in the body. In addition, examples of hormones produced by the anterior pituitary glad upon stimulation with gonadotropin releasing hormone include follicle-stimulating hormone and luteinizing hormones. These hormones stimulate the gonads, such as the ovaries and the testes, to secrete androgens, such as testosterone, progesterone, and estrogen, which in turn affect sexual development, sexual behavior, and other reproductive and nonreproductive functions. As a result, the endogenous opioid system plays an important role in modulating endocrine, reproductive, cardiovascular, respiratory, gastrointestinal, immune functions, sexual development and function, as well as a person's response to stress.
More specifically, in humans, it has been determined that chronic administration of opioids has an inhibitory effect on the HPA axis [McDonald et al., Effect of morphine and nalorphine on plasma hydrocortisone levels in man. J. Pharmacol. Exp. Ther. 125:241247 (1959)]. Basal levels of ACTH and cortisol are significantly disrupted in active heroin addicts: suppression of ACTH and cortisol and abnormal diurnal rhythms of these hormones are found [Kreek, Medical safety and side effects of methadone in tolerant individuals. JAMA 223:665–668 (1973)]. Basal levels and the diurnal rhythm of ACTH and cortisol, which are disrupted in active heroin addicts, have been shown to become normalized in moderate to high dose, long-term methadone-maintained patients when compared to those of healthy volunteer subjects [Kreek, 1973; Kreek et al., Circadian rhythms and levels of beta-endorphin, ACTH, and cortisol during chronic methadone maintenance treatment in humans. Life Sci. 33:409–411 (1983); Kreek et al., Prolonged (24 hour) infusion of the opioid antagonist naloxone does not significantly alter plasma levels of cortisol and ACTH in humans. Proceedings of the 7th International Congress on Endocrinology, Elsevier Science, p. 1170, 1984].
In healthy volunteers, ACTH and cortisol levels decrease below the basal levels in response to the infusion of β-endorphin indicating feedback of inhibition of pituitary ACTH release or suppression of hypothalamic CRF release by β-endorphin [Taylor et al., Beta-endorphin suppresses adrenocroticotropin and cortisol levels in normal human subjects. J. Clin. Endocrinol. Metab. 57:592–596 (1983)], and also naloxone (an opioid antagonist) stimulates a rise in serum ACTH and cortisol, suggesting that the HPA axis is under the tonic inhibitory control of endogenous opioids normalized in steady-state chronic methadone-maintained patients; their HPA axis responses to metyrapone-induced stress appear to be no different from that of healthy volunteer subjects [Kreek, 1973; Kreek et al., Prolonged (24 hour) infusion of the opioid antagonist naloxone does not significantly alter plasma levels of cortisol and ACTH in humans. Proceedings of the 7th International Congress on Endocrinology Elsevier Science, p. 1170, 1984].
Support for the effects of opioids on physiological functions regulated by the HPA and the HPG axes can be found in observations of heroin addicts. More specifically, it has been observed that many heroin addicts are infertile, and in the case of female addicts, their menstrual cycle is dramatically disrupted to the point that they do not ovulate. Furthermore, it has been observed that heroin addicts, and nonaddicted patients taking morphine, become constipated, and that the immune systems of addicts is weakened relative to the immune system of non addicts. However, once therapeutic agents designed to treat addiction, such as methadone, addicts become fertile, are no longer constipated, and have a immune system whose ability to fight foreign bodies is in parity with the immune system of a nonaddict.
Hence, what is needed is discovery of additional, heretofore unknown polymorphisms of the human mu opioid receptor gene that can be used as genetic markers to map the locus of the human mu opioid receptor gene in the genome.
What is also needed are the DNA sequences of heretofore unknown isolated nucleic acid molecules which encode human mu opioid receptors, wherein the DNA sequences include a combination of presently known and subsequently discovered polymorphisms of the human mu opioid receptors.
Furthermore, what is needed is the characterization of the binding properties of heretofore unknown human mu opioid receptors produced from the expression of genes comprising such heretofore unknown polymorphisms of the human mu opioid receptor gene, or combinations of unknown polymorphisms and known polymorphisms.
Furthermore, what is needed is a characterization of the activity of such unknown human mu opioid receptors produced from the expression of nucleic acid molecules comprising such polymorphisms.
What is also needed is a correlation between polymorphisms of the human mu opioid receptor gene, and the susceptibility of a subject to addictive diseases, such as heroin addiction, cocaine addiction, or alcohol addiction, to name only a few.
What is also needed are diagnostic methods to determine a subject's increased or decreased susceptibility to addictive diseases. With the results of such methods, targeted prevention methods, early therapeutic intervention, and improved chronic treatment to opioid addiction can be developed. Physicians armed with the results of such diagnostic methods can determine whether administration to a subject of opioid analgesics is appropriate or whether non-opioid derived analgesics should be administered to the subject. Also, appropriate choice and type of analgesic can be made in treating a subject's pain.
What is also need are methods of determining a subject's susceptibility to pain and responsibility to analgesics, and using that information when prescribing analgesics to the subject.
What is also needed is an ability to determine the binding affinity of the mu opioid receptor to endogenous opioids, such as β-endorphin, and the effect of this binding activity on the neuroendocrine system.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.